Method for detecting cancer and tumor invasiveness using dna palindromes as a biomarker

Abstract

Disclosed herein are a method of detecting invasive tumor in a subject and a method of treating a subject with cancer based on invasiveness of tumor. In various embodiments, the method of detecting tumor invasiveness includes denaturing genomic DNA isolated from a tumor sample obtained from the subject; renaturing the denatured DNA for tumor DNA palindrome to form a snap back DNA; digesting the renatured DNA with a nuclease that digests single strand DNA; amplifying the tumor DNA palindrome by adapter ligation-mediated polymerase chain reaction (PCR) with genome-wide analysis of Palindrome Formation (GAPF); performing a sequence scan across multiple samples of the amplified tumor DNA palindrome; mapping reads of GAPF-seq from the sequence scan into a plurality of bins; quantifying reads in each bin; and determining whether the tumor sample is an invasive tumor based on GAPF profiles generated by analyzing the quantified reads in each bin.

1 . A method of detecting invasive tumor in a subject, the method comprising:
 denaturing genomic DNA isolated from a tumor sample obtained from the subject or denaturing cell-free DNA (cfDNA) isolated from a body fluid obtained from the subject having the tumor, to generate denatured DNA;   renaturing the denatured DNA for tumor DNA palindrome to form a snap back DNA;   digesting the renatured DNA with a nuclease that digests single strand DNA;   amplifying the tumor DNA palindrome by adapter ligation-mediated polymerase chain reaction (PCR) with genome-wide analysis of Palindrome Formation (GAPF);   performing a sequence scan across multiple samples of the amplified tumor DNA palindrome;   mapping reads of GAPF-seq from the sequence scan into a plurality of bins;   quantifying reads in each bin; and   determining whether the tumor sample is an invasive tumor based on presence of tumor-derived DNA in the genomic DNA or cfDNA and/or GAPF profiles generated by analyzing the quantified reads in each bin.   
     
     
         2 . The method of  claim 1 , wherein:
 the subject is determined to have the invasive tumor when the tumor sample or body fluid is GAPF-positive or when any one of chromosomes has more than a threshold number of bins out of top 1,000 bins; or   the subject is determined to not have an invasive tumor when the tumor sample or body fluid is GAPF-negative or when no tumor DNA palindromes are detected from the isolated genomic DNA or cfDNA.   
     
     
         3 . The method of  claim 2 , wherein the determination is based on a chromosome-specific threshold. 
     
     
         4 . The method of  claim 1 , wherein the tumor is stage I tumor or luminal A tumor. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 4 , wherein the tumor DNA palindrome clusters at CCND1 oncogene loci in the luminal A tumor. 
     
     
         7 . The method of  claim 1 , wherein the subject has breast cancer or lung cancer. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein numbers of the GAPF-seq reads are counted for 1-kb bins. 
     
     
         10 . The method of  claim 9 , wherein top 1,000 bins are taken for analysis to determine the presence of the invasive tumor. 
     
     
         11 . The method of  claim 1 , wherein the body fluid of the subject comprises interstitial fluid, intravascular fluid, transcellular fluid, amniotic fluid, aqueous humor, bile, blood, whole blood, blood serum, blood plasma, breast milk, cerebrospinal fluid, cerumen, chyle, exudates, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine, or vomit. 
     
     
         12 . The method of  claim 11 , wherein the body fluid comprises blood or blood plasma. 
     
     
         13 . The method of  claim 1 , wherein the sequence scan is a shallow scan, and the method does not require deep sequencing. 
     
     
         14 . The method of  claim 1 , wherein an amount of the genomic DNA required for generation of the GAPF profiles is about 10 ng-about 50 ng, about 20 ng-about 40 ng, about 25 ng-about 35 ng, or about 30 ng or less. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1 , further comprising isolating the genomic DNA from the tumor before denaturing the genomic DNA. 
     
     
         19 . The method of  claim 1 , further comprising isolating the cf DNA from the body fluid before denaturing the cfDNA. 
     
     
         20 . (canceled) 
     
     
         21 . A method of treating a subject with cancer based on invasiveness of tumor, the method comprising:
 administering a treatment to the subject, the treatment comprising biopsy, surgery, chemotherapy, hormone therapy, and/or radiation therapy if the tumor is an invasive tumor, or the treatment comprising active monitoring, not performing biopsy, surgery, chemotherapy, hormone therapy, and radiation therapy on the subject, if the tumor is not an invasive tumor,   wherein tumor invasiveness is detected by:   denaturing genomic DNA isolated from a tumor sample obtained from the subject or denaturing cell-free DNA (cfDNA) isolated from body fluid obtained from the subject having the tumor, to generate denatured DNA;   renaturing the denatured DNA for tumor DNA palindrome to form a snap back DNA;   digesting the renatured DNA with a nuclease that digests single strand DNA;   amplifying the tumor DNA palindrome by adapter ligation-mediated polymerase chain reaction (PCR) with genome-wide analysis of Palindrome Formation (GAPF);   performing a sequence scan across multiple samples of the amplified tumor DNA palindrome;   mapping reads of GAPF-seq from the sequence scan into a plurality of bins;   quantifying reads in each bin; and   determining the invasiveness of the tumor in the subject based on presence of tumor-derived DNA in the genomic DNA or cfDNA and/or GAPF profiles generated by analyzing the quantified reads in each bin.   
     
     
         22 . The method of  claim 21 , wherein the cancer comprises breast, prostate, or lung cancer. 
     
     
         23 . The method of  claim 22 , wherein the treatment of the breast cancer comprises surgery, radiation, chemotherapy, hormone therapy, targeted drug therapy, and/or immunotherapy based on a stage/type of the breast cancer if the tumor is an invasive tumor. 
     
     
         24 . The method of  claim 22 , wherein the treatment of the lung cancer comprises surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, palliative care, and/or alternative medicine such as acupuncture, hypnosis, massage, meditation, and yoga based on a stage/type of the lung cancer if the tumor is an invasive tumor. 
     
     
         25 . The method of  claim 22 , wherein the treatment of the prostate cancer comprises surgery, radiation, cryotherapy, hormone therapy, chemotherapy, immunotherapy, and/or targeted drug therapy based on a stage/type of the prostate cancer if the tumor is an invasive tumor. 
     
     
         26 . The method of  claim 21 , wherein the body fluid of the subject comprises interstitial fluid, intravascular fluid, transcellular fluid, amniotic fluid, aqueous humor, bile, blood, whole blood, blood serum, blood plasma, breast milk, cerebrospinal fluid, cerumen, chyle, exudates, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine, or vomit. 
     
     
         27 . The method of  claim 26 , wherein the body fluid comprises blood or blood plasma. 
     
     
         28 . (canceled)